Metal surface, fluorescence enhancement

In addition to the field enhancement, the increases of the radiative decay rate of the molecule also lead to the fluorescence enhancement. This happens when molecules are S -20nm away from metal nanoparticies aggregated on surfaces [19-21]. Lakowicz and coworkers have characterized this phenomena by using silver island films deposited on the internal surface of two quartz plates which sandwich a bulk fluorophore solution [20]. The fluorophores are physically placed close to silver islands so that there are a range of distances between the fluorophore and metal. The fluorescence enhancement is accompanied by decreased lifetimes and increased photostability. This phenomenon shows that the silver island increases the radiative decay rate of the fluorophore and therefore induces the fluorescence enhancement. 

The geometry of the nanoscaled metals has an effect on the fluorescence enhancement. Theoretically, when the metal is introduced to the nanostructure, the total radiative decay rate will be written as T + rm, where Tm corresponds to the radiative decay rate close to the metal surface. So, (1) and (2) should be modified and the quantum yield and lifetime are represented as ... 

Since the pioneering work by Cotton et al. on heme proteins (Cotton et al., 1980), surface enhanced resonance Raman spectroscopy (SERRS), Sec. 6.1, has been used to study a large variety of biomolecules, such as retinal proteins (Nabiev et al., 1985), flavoproteins (Coperland et al., 1984 Holt and Cotton, 1987), chlorophylls (Cotton and Van Duyne, 1982 Hildebrandt and Spiro, 1988), and oxyhemoglobins (de Groot and Hesters, 1987). The advantages of this technique include low sample concentration and fluorescence quenching. The main question is whether or not the native structure and function of the molecule is preserved on the metal surface. 

Figure 1.2. (A) A schematic diagram depicting the processes in close proximity to metals (< 10 nm) involved in Metal-Enhanced Fluorescence enhanced absorption and coupling to surface piasmons. (B) Emission spectra of FITC deposited onto SIFs and glass. The inset shows the real-color photographs of FITC emission from these surfaces. (C) Intensity decays for FITC on both glass and SiFs. IRF Instrument Response Function.

When a fluorophore is located in close proximity to a metal surface, both its absorption and emission properties may be affected dramatically. This in turn affects its fluorescence properties and may result in either a quenching or an enhancement of the fluorescence signal. This latter situation is obviously of interest for many applications using fluorophores. Let us discuss these steps in more detail. 

In conclusion, the fluorescence signal is not necessarily completely quenched for molecules directly adsorbed on the metal surface, but it is rather much less enhanced than the Raman signal. As a consequence, if SERS peaks can be observed for a fluorophore, they should in most cases be accompanied by a MEF signal. 

In the experiments presented here, the fluorophores are adsorbed directly onto the metallic (gold) NP surface. The fluorescence enhancement (if any) is therefore small and the MEF signal may accordingly be weak, especially for weak fluorophores like Rh6G at 633 nm. In fact, the MEF spectrum is accompanied by Raman peaks of comparable intensity (themselves enhanced through Surface Enhanced Raman Scattering, SERS). 

Although it has been difficult to separate the effects of excitation and emission enhancement, both of these effects should be extremely sensitive functions of the shape of the metal particle, the orientation of the fluorophore, and the distance between the fluorophore and the metal, because the local-field effects depend strongly on these parameters. Many groups have studied variations in fluorescence intensity as a function of the distance between a layer of fluorophores and a number of nanostructured metal surfaces, adsorbed colloidal particles or suspended colloidal particles. Single-molecule experiments have even provided strong evidence for the existence of a local maximum in the fluorescence intensity versus distance curve. ... 

In order to establish that the enhancement was due to the LSPR effect and not to variations in the dye emission when conjugated to the silica shell surface, a separate experiment was performed where the metal NP was replaced by a pure silica NP with the same radius. These NPs were synthesized using a microemulsion technique [18] and the dye was attached as in the case of the metal NPs. The enhancement measurement was repeated and the fluorescence from the dye - silica NP was almost identical to that measured in solution hence confirming the plasmonic nature of the enhancement. 

The fluorescence amplification provided by the plasmonic nanostructures has been shown to be applicable to many fluorophores. Hence fluorophores currently employed in assays would still be suitable. However, the use of low quantum yield fluorophores would lead to much larger fluorescence enhancements (i.e. 1 / Qo) and could significantly reduce unwanted background emission fi om fluorophores distal fi om the metallic surface. 

Shape The radiative emission from molecules confined within metallic nanocavities and on the surface of nanoparticles is of great relevance to biotechnology. In 1986, it has been suggested that fluorescence enhancement and reduced observation volumes could be obtained from small metal apertures (85). Nanocavities of different shapes could induce different surface plasmon (SP) fields. More recently, some studies has been done for different shapes, such as circular (86-90), elliptical (91), coaxial (92), or rectangular (93, 94) metallic nanocavity(95). In 2003, single-molecule detection from a nanocavity was demonstrated (86). However, it might be difficult to position the biospecies in the nanocavities. 

Several other studies (150-153) reported that metal surfaces were able to either enhance or suppress the radiative decay rates of fluorophores. Furthermore, an increase in the extent of resonance energy transfer was also observed. These effects might be due to the interactions of excited-state fluorophores with SPs, which in turn produce constructive effects on the fluorophore. The effects of metallic surfaces include fluorophore quenching at short distances ( 0-5 nm), spatial variation of the incident light field (-0-15 nm), and changes in the radiative decay rates (-0-20 nm) (64). The term of metal-enhanced fluorescence could be referred to the appplication of fluorophore and metal interactions in biomedical diagnosis (64). 

It is possible that surface enhancement effects, similar to the observations made earlier in metal-fluorophore systems [11, 83-85] may occur. Metal surfaces are known to have effects on fluorophores such as increasing or decreasing rates of radiative decay or resonance energy transfer. A similar effect may take place in ZnO nanomaterial platforms. However, decay lengths of fluorescence enhancement observed in the semiconducting ZnO NRs are not commensurate with the length scale seen on metals such as Au or Ag. For effective metal enhanced fluorescence, fluorophores should be placed approximately between 5-20 nm away from the metal surface. However, fluorescence enhancement effect on ZnO NRs is observed even when fluorophores are located well beyond 20 nm away from the NR surface. At the same time, no quenching effec en when they are placed directly onto ZnO NR surfaces. In addition, there overlap between the absorption and emission... 

Figure 16.3 Modified Jablonski diagram ows the energy absorption effects of near metal surface enhanced fluorescence. The process involves o eating an excited electronic singlet state by optical absorption and subsequent emission of fluorescence with different decay paths.

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